Characterization of a rabbit skeletal muscle protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin.
A protein kinase (designated PC0.7 in DePaoli-Roach, A. A., Roach, P. J., and Larner, J. (1979) J. Biol. Chem. 254, 12062-12068) that phosphorylated both glycogen synthase and phosvitin, has been extensively purified from rabbit skeletal muscle, close to apparent homogeneity. The enzyme activity was associated with two polypeptides, alpha (Mr = 43,000) and beta (Mr = 25,000), present in approximately equimolar amounts. The apparent molecular weight of the enzyme was 180,000, as determined by gel filtration, and 130,000, as judged from sucrose density gradient sedimentation. Unless precautions were taken during the purification, the alpha polypeptide underwent degradation, probably as a result of protease action. The beta polypeptide itself could be phosphorylated upon incubation of the enzyme with ATP and Mg2+ but no significant change in activity accompanied this phosphorylation reaction. The protein kinase was effective in utilizing both ATP and GTP as phosphate donors, with apparent Km values of 13 microM and 20-35 microM, respectively. The apparent Km values for phosvitin and glycogen synthase were 15 microM and greater than 10 microM, respectively. PC0.7 phosphorylated glycogen synthase to a level of approximately 0.5 phosphate/subunit, with little inactivation of the glycogen synthase. Phosphorylation occurred predominantly in a 21,000-dalton cyanogen bromide fragment of glycogen synthase, the same fragment preferentially phosphorylated by cyclic AMP-dependent protein kinase. This phosphorylation was also located in an approximately 17,000-dalton COOH-terminal region of the glycogen synthase molecule that is removed by limited tryptic proteolysis. Phosphorylation of glycogen synthase by PC0.7 occurred at serine residues whereas in phosvitin both serine and threonine residues were modified by PC0.7 action.
D000818:Animals; D002846:Chromatography, Affinity; D004527:Egg Proteins / Q000378:metabolism*; D006006:Glycogen Synthase / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010774:Phosvitin / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D004527:Egg Proteins; D010446:Peptide Fragments; D010774:Phosvitin; D006006:Glycogen Synthase; D011494:Protein Kinases